The present invention relates to an apparatus for conducting separation of biological materials, such as peptides, proteins, cells, cell organelles, cell membranes, viruses, and organic and inorganic compounds or all metal ions from a mixture. More particularly, the present invention relates to an apparatus and method for batch or continuous-flow electrophoretic separation of ionic molecules by zone electrophoresis, isoelectric focusing, counterflow gradient focusing or by electrodialysis.
Electrophoresis is an analytical and preparative tool which can separate components of a mixture on the basis of their ionic charge and mass. A mixture of ionic species is exposed to an applied voltage field, which causes the ions to migrate toward the oppositely charged electrode at a rate which depends on their electrophoretic mobility, which in turn depends on charge, mass and symmetry as well as other parameters. Different modalities of electrophoresis, e.g., zone, isoelectric focusing, isotachophoresis, moving boundary can be utilized in various instruments that are classified by the method selected to minimize convective mixing: 1) free fluid methods which use thin film, shear, capillaries, or compartmentation; or 2) supported fluid methods which use density gradient gels or other matrices. The present method is free fluid compartmentation.
Zone electrophoresis is the separation carried out in the presence of a homogenous buffer, in which sample components separate according to their mobility. No steady state is achieved, and migration continues with gradual broadening of the sample zones due to diffusion and other effects.
Isoelectric focusing (IEF) is a variant based upon the fact that most biomaterials are amphoteric in nature, i.e., are positively charges in an acidic environment and negatively charged in a basic environment. At a particular pH value, called the isoelectric point (pI), the biomaterials acquire a zero net charge due to the balance of positive and negative charges. When such amphoteric materials are exposed to an applied electrical field, in a medium exhibiting a pH gradient, they will migrate toward the pH region of their pI and become immobilized or focused in a steady state at that pH region.
Electrophoretic separation in multi-compartment electrolyzers has been known since 1912 (Ikeda et al, U.S. Pat. No. 1,015,891) when it was introduced for preparative scale protein fractionation. To the present, the basic apparatus has undergone multiple changes and has been improved in many ways.
Current designs feature membrane-separated, multicompartment electrophoretic cells with cylindrical, annular or rectangular cross-sections, and membrane-separated electrode compartments. The separation process is usually carried out in the free solution under batch process conditions. The entire process volume of the solution may be contained within the electrophoretic cell during the separation process, or the process volume of the solution may be recirculated through the electrophoretic cell until completion of the separation process.
Several electrophoretic devices for isoelectric focusing have been designed by Rilbe, with the latest improved version published in 1980. This latest device consists of a rotating cylinder with 46 subcompartments, each 1.5 cm wide and 12.5 cm in diameter, and has a total volume of 7.6 liters. The subcompartments are separated by polyvinylchloride (PVC) membranes, while, at the ends, one of the electrode compartments is separated by cellophane and the other by PVC. The cooling of the solution is achieved by a combination of inner and outer cooled surfaces. The cylinder is submerged in 360 liters of refrigerated water and rotated about the separation axis, thereby cooling the cylinder walls. Also, four glass cooling tubes pass through each subcompartment and, when the cylinder is rotated about its longitudinal axis, water is scooped into one side of the cooling tube and forced out the other.
While the internal cooling system is a beneficial feature of the apparatus, the choice of materials for the membranes and the excessive width of the subcompartments are disadvantageous. PVC exhibits a 5-10 fold greater electroosmotic effect than a woven monofilament nylon screen. Electroosmotic mixing is also increased by the small porosity of the PVC membranes, which trap colloidal and larger protein particles. PVC also binds proteins with considerable affinity. Protein precipitations at a position in the pH gradient, deviating from the respective pI, produce a net charge on the immobilized protein resulting in electroosmotic mixing. By using the chosen membrane materials, i.e., cellophane and PVC, electroosmotic transport across the electrode membrane occurs, which induces mixing. Electroosmotic transport counteracts and degrades the separation process. The large width of each subcompartment also retards the separation process. Also, the low ionic strength electrolytes, which are employed in Rilbe's apparatus, cause a voltage drop in the electrode chambers, which decreases the effective field strength in the separation compartments.
Another typical device for electrophoretic separation processes is disclosed in U.S. Pat. No. 4,588,492 issued May 13, 1986 to Bier. This apparatus, known by the trademark ROTOFOR, is essentially a smaller version of the apparatus by Rilbe. It consists of twenty annular-shaped compartments, which are arranged into a cylindrical electrophoretic cell. The individual compartments are separated by membranes, and an internal cooling tube is arranged along the longitudinal axis of the cell. The cell is usually operated in a horizontal position while rotated along its longitudinal axis. It has recently been demonstrated by Egen, N. B., et al., "Fluid Stabilization Studies of Free IEF in Cylindrical and Annular Columns" Separation Science & Technology, Vol. 22, p. 1383 (1987), however, that equal separation results could be achieved in a vertical stationary mode while minimal separation was observed in a horizontal stationary operation mode. The ROTOFOR eliminates some of the drawbacks of Rilbe's device by using woven nylon screen, small width subcompartments and ion exchange membranes to separate the electrode compartments. The disadvantage of the ROTOFOR is that, due to the annular cross section and the central inner cooling tube, scale-up to significantly larger capacities is impossible, since an increase of the cylinder diameter would result in an insufficient cooling of the solution close to the outer walls of the cylinder. The ROTOFOR is strictly a batch mode processor and cannot be operated in a continuous flow mode.
Multi-compartment electrolyzers which separate proteins by the principles of IEF are also known. For example, U.S. Pat. Nos. 4,362,613 and 4,204,929, both issued to M. Bier on Dec. 7, 1982 and May 27, 1980, respectively, disclose such apparatus. However, this is designed to operate exclusively by IEF and in the batch mode, whereby the solution is recycled between the electrolytic cell and an external heat exchanger to provide external cooling of the sample solution.
A method for the continuous-flow electrophoretic separation of proteins, antibodies, nucleic acids etc. in a matrix system is known and is shown in U.S. Pat. No. 4,323,439 issued to P. O'Farrell on Apr. 6, 1982. Matrices of the size-exclusion type, of the ionic type, of the adsorption-type or of a special affinity to the particles to be separated are used. The separation chamber comprises at least one matrix of continuously varying properties or two and more matrices of continuously or intermittently varying properties in the longitudinal direction. The mixture to be separated is introduced into the separation chamber in a carrier liquid which flows through the length of the chamber. An electric field is applied such that the electrophoretic movement occurs in a direction opposite to the carrier liquid flow. By countering the electrophoretic movement with counter-current flow, an equilibrium zone within the chamber is created, where the separated and concentrated ions may be withdrawn. However, matrices are expensive and the random withdrawal of the separated and concentrated ionic species will cause flow discontinuities. An electrophoretic separator in a continuous flow, free solution mode is highly desirable.